Molecular chaperons are proteins which mediate protein folding. They bind non-covalently to exposed surfaces of proteins that are newly synthesized or are denatured or misfolded and assist them to fold into correct conformations. Molecular chaperons are also involved in a number of cellular processes such as protein synthesis protein translocation and DNA replication.
Molecular chaperons include heat shock proteins, which are proteins whose expression increases significantly in cells following an exposure to unusually high temperature (heat shock) or an exposure to a wide variety of physiological stresses. This increase in the molecular chaperon expression in turn provides cells with protection against the adverse effects of hyperthermia as demonstrated by the thermotolerance of cells for otherwise lethal temperatures if the cells are preconditioned by a brief exposure to high temperature.
Physiological stresses inducing heat shock protein expression include a wide variety of pathological conditions associated with many diseases. The synthesis of heat shock proteins in cells exposed to such stresses, indicates the protection of the cell against the physiological stresses, like also in the case of the heat shock response
One such pathological condition associated with induction of molecular chaperons is ischemic injury. Ischemic injury to tissues results from deterioration of blood supply for any possible. For instance, prolonged coronary occlusion causes severe damage to myocardium, leading to myocardial necrosis and jeopardizing the chances for recovery even if the blood flow is restored. In brain, to significant damages may frequently be caused by ischemia, leading to death of the brain-tissue.
It was observed that the amount of heat shock protein hsp70 increased in the myocardium during ischemia leading to necrosis even if the duration of ischemia is short. In these cases, likewise in a heat shock, the enhanced hsp70 content of the cells protects the same against the consequences of a next ischemia, which would otherwise cause necrosis (DAS, D. K. et al. Cardiovascular Res.: 578. 1993). It has also been observed when rat cells in culture were subjected to ischemia, J. Clin. Invest., 93: 759-767 (1994)). Accordingly, heat shock proteins synthesized by myocardial cells provide protection against ischemic injuries.
The situation in brain-tissue is similar, wherein cerebral ischemia results in increased expression of heat shock protein in the brain tissue. Experiments have also proved that pretreatment of animals with sub-lethal ischemia induces heat stress protein (hsp70) and protects the brain against more severe subsequent ischemic insult. (Simon, et al., Neurosci. Lett., 163:135-137 (1993)).
Yet another example of physiological stress on tissues and organs associated with molecular chaperon induction is provided by inflammatory diseases. Inflammation is a non-specific response of host cells to entry of foreign material, such as in case of infection by various bacterial and viral pathogens, and involves aggregation and activation of leukocytes to the injury site, which results in production and release of high levels of reactive oxygen species and cytokines. These cytokines and reactive oxygen radicals attack the pathogen, but also damage the host tissues (Jaquier, Sarlin, Experientia, 50: 1031-1038/19940). It is believed that as a protection against these toxic mediators of inflammation, the host tissues increase production of molecular chaperons. Molecular chaperons thus produced protect host cells from damages caused by reactive oxygen species and protect cells from cytotoxicity of TNF and other cytokines and reactive oxygen radicals. In animal studies, it has been demonstrated the pre-exposure of an animal to heat shock, with resulting increase of a heat shock protein (hsp70) expression, resulted in remarkable decrease in pulmonary inflammation. Accordingly, molecular chaperons serve anti-inflammatory function.
The above examples illustrate ability of molecular chaperons to protect cells against various physiological stresses disturbing cellular homeostatic balance and causing injury to cells. Molecular chaperons have also been shown to be advantageous in treating neoplasms. For example, it has been reported that when tumor cells are transfixed with a gene encoding a molecular chaperon (65 kd hsp), they lose or show decrease in their tumorigenicity (PCT Application No. PCT/GB93/02339). Furthermore, it has also been reported that tumor cells, in response to heat stress, express molecular chaperons in increased amount. However, they are present not in cytoplasm, but on the surface of cell membranes. (Ferrarini, M. et al. Int. J. Cancer, 51:613-619/1992/). Increased presence of molecular chaperons on cell surfaces correlates with increased sensitivity of NK (natural killer) cells toward the tumor cells, allowing better targeting, infiltrating, and killing of the tumor cells by NK cells (Kurosawa, S. et al. Eur. J. Immunol. 23:1029/1993/).
In view of the advantages associated with increased molecular chaperon expression in cells, a method which increased such expression or increased activity of molecular chaperons would be highly desirable.
The invention relates to methods for increasing expression or enhancing activity of molecular chaperons by a cell. In particular, according to one non-limiting embodiment of the invention, a method is provided comprising treating a cell that is exposed to a physiological stress with an effective amount of a chemical compound during, before or after the physiological stress which increases expression of a molecular chaperon in the cell beyond the amount induced by the physiological stress,
wherein the chemical compound is a hydroxylamine derivative the tautomeric forms of which are represented by formulae (I) and (II), or its salt, including the optically active stereoisomers thereof, wherein
A is an alkyl, substituted alkyl, aralkyl, aralkyl substituted in the aryl and/or in the alkyl moiety, aryl, substituted aryl, heteroaryl or substituted heteroaryl group,
Z is a covalent bond, oxygen or xe2x95x90NR3 wherein R3 is selected from the group consisting of hydrogen, an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or aralkyl substituted in the aryl and/or in the alkyl moiety,
R is an alkyl or substituted alkyl,
X in the tautomer of formula (I) is halogen or a substituted hydroxy or amino, monosubstituted amino or disubstituted amino group and
X in the tautomer of formula (II) is oxygen, imino or substituted imino group and
Rxe2x80x2 is hydrogen, an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, aralkyl having substituted aryl and/or alkyl moiety, acyl or substituted acyl group,
and the compounds of formula (I) optionally contain intramolecular ring structures formed by coupling X and a reactive substituent.
An other non-limiting embodiment of the invention is the method of enhancing the activity of a molecular chaperon in a cell exposed to a physiological stress which comprises administering an effective amount of a hydroxylamine derivative of structure (I) or (II), as described above. Thus, the activity of molecular chaperon is increased beyond the amount induced by the physiological stress alone. In either of these methods, it is preferred that the cell to which the hydroxylamine derivative is administered to is an eukaryotic cell.
According to the invention eucaryotic cells are treated with the hydroxylamine derivatives as defined above.
Another object of the invention is the method of treatment, or possible prevention of diseases connected with functioning of the chaperon system or associated with damages of the cell- or cell-organellum membrane, wherein for suppressing the pathological condition effective amount of a hydroxylamine derivative of the formula (I) or (II) is administered to the host organism.
Still another object of the invention is the use of the hydroxylamine derivatives of the formula (I) or (II) or the salts thereof in the preparation of pharmaceutical compositions which can be used in the treatment of cardiovascular, vascular, cerebral, tumorous diseases, diseases of the skin and/or mucous membrane or those of the epithelial cells of renal tubules, as well as in the preparation of cosmetical compositions.
The invention further relates to novel hydroxylamine derivatives possessing a wide range of biological effect and are useful for enhancing the level of molecular chaperon in organisms or the activity of the said molecular chaperons and for the preparation of pharmaceutical and cosmetical compositions applicable to this purpose.
A further object of the invention is represented by the pharmaceutical and cosmetical compositions which comprise novel hydroxylamine derivatives together with carriers and auxiliaries generally acceptable in such compositions.
The present invention is based, at least in part, on an unexpected discover, that hydroxylamine derivatives having structures as described above, when used in the treatment of cells, are capable of increasing the amount of molecular chaperons produced by that cell or enhancing the activity thereof. This effect is particularly great when the cell is under physiological stress which induces molecular chaperon expression. In such cases, the chemical compound enhances expression of molecular chaperons by the cell beyond that amount induced by the physiological stress alone. This discovers is significant in view of the role molecular chaperons play in cells defending themselves against pathological effects of various diseases. Thus, if a compound is able to increase the amount or enhance the activity of molecular chaperons being expressed by cells, this allows the cells to be protected against the deleterious effects of the diseases and to repair damages caused by them.
FIG. 1 shows the changes in hsp level on H9c2 rat myocardium exposed to heat shock by the effect of treatment with N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloride maleate. This compound is labeled B on the Figures and referred to as compound B in the followings as well.
FIG. 2 shows the results of the above experiment obtained by Western blot analysis based on densitometric evaluation.
FIG. 3 shows the results of hsp70 mRNA Northern blot analysis obtained during examination of the effect of compound B on cellular hsp expression at transcription level.
FIG. 4 shows the results of hsp26 mRNA Northern blot analysis obtained on Saccharomyces cerevisiae cells during examination of the effect of compound B on hsp activation.
FIG. 5 shows the effect of benzyl alcohol on the adenylate cyclate activation and the membrane state of plasma.
FIG. 6 shows the hsp gene expression rate on HeLa cells, using luciferase reporter gene for the test.
FIG. 7 illustrates the effect of compound B on hsp72 cell surface expression in K562 cell line.
FIG. 8 shows the interaction of compound B and different lipid membranes showing the increase of surface pressure.
FIG. 9 shows the effect of compound B in the concentration of 10 mM and 100 mM on the bilayer (La)xe2x86x92T hexagonal (HH) phase transfer of large unilamellar vesicules prepared from dipalmitoyl-phosphatidyl ethanolamine.
FIG. 10 is the diagram of the effect of compound B on the serum TNF level in healthy and STZ diabetic rats.
FIG. 11 shows the effect of compound B against the growth inhibiting effect of keratinocyte cyclohexylimide.
FIG. 12 shows the effect of compound B against the cell damaging effect of cycloheximide on endothelial cells.
FIG. 13 shows the cytoprotective effect of compound B against the cell damaging effect of cycloheximide on HeLa cell line.
FIG. 14 shows the effect of compound B against the growth inhibiting effect of cyclohexylimide on H9c2 rat myocardium cell line.
FIG. 15 shows the effect of compound B on the P1 transcription factor activity in AB 1380 yeast cells.
FIG. 16 shows the effect of compound B on the AP1 transcription factor activity in JF1 yeast cells.
FIG. 17 shows the effect of compound B on the P1 transcription factor activity in AB 1380 yeast cells.
FIG. 18 shows the test results obtained on isolated functioning ischemic rat heart model wherein the model was treated with compound B, determined by Western blotting 2 hours after ischemia.
FIG. 19 shows the test results obtained on isolated functioning ischemic rat heart model wherein the model was treated with compound B, determined by Western blotting 3 hours after ischemia.
FIG. 20 shows the wound healing on STZ diabetic rats after heat injury by treatment of cream containing 1% compound B.
FIG. 21 shows the wound healing on STZ diabetic rats after heat injury by treatment of cream containing 2% compound B.
FIG. 22 shows the wound healing on STZ diabetic rats after heat injury by treatment of cream containing 4% compound B.
FIG. 23 shows the wound healing on STZ diabetic rats after heat injury by treatment of cream containing 1% compound B, but evaluated visually.
FIG. 24 shows the wound healing on STZ diabetic rats after heat injury by treatment of cream containing 2% compound B, but evaluated visually.
FIG. 25 shows the wound healing on STZ diabetic rats after heat injury by treatment of cream containing 4% compound B, but evaluated visually.
FIG. 26 shows the comparison photographs (treated and control) made in the above tests by digital epiluminescence microscopic technique.
FIG. 27 shows the hsp72 level of the samples obtained in the previous tests determined by Western blotting, at the treatments with creams containing 1, 2 and 4% compound B.
FIG. 28 shows the hsp72 levels determined by immunohistochemical analysis (treated and control) on SCID mice exposed to UV-B ray and treated with compound B.
FIG. 29 shows the hsp72 levels determined by Western blotting on SCID mice skin biopsy samples exposed to UV-B ray and treated with compound B.